Expression and functional validation of heat-labile enterotoxin B (LTB) and cholera toxin B (CTB) subunits in transgenic rice (Oryza sativa)

Abstract

We expressed the heat-labile enterotoxin B (LTB) subunit from enterotoxigenic Escherichia coli and the cholera toxin B (CTB) subunit from Vibrio cholerae under the control of the rice (Oryza sativa) globulin (Glb) promoter. Binding of recombinant LTB and CTB proteins was confirmed based on GM1-ganglioside binding enzyme-linked immunosorbent assays (GM1-ELISA). Real-time PCR of three generations (T3, T4, and T5) in homozygous lines (LCI-11) showed single copies of LTB, CTB, bar and Tnos. LTB and CTB proteins in rice transgenic lines were detected by Western blot analysis. Immunogenicity trials of rice-derived CTB and LTB antigens were evaluated through oral and intraperitoneal administration in mice, respectively. The results revealed that LTB- and CTB-specific IgG levels were enhanced in the sera of intraperitoneally immunized mice. Similarly, the toxin-neutralizing activity of CTB and LTB in serum of orally immunized mice was associated with elevated levels of both IgG and IgA. The results of the present study suggest that the combined expression of CTB and LTB proteins can be utilized to produce vaccines against enterotoxigenic strains of Escherichia coli and Vibrio cholera, for the prevention of diarrhea.

Keywords: Enterotoxin; Immunogenicity; Oral vaccine; Oryza sativa; Transgenic plant.

Figure 1

Schematic representation of the binary vector containing LTB and CTB. Glb, Glb promoter (0.9 kb); TpinII, potato protease inhibitor II terminator (1.0 kb); P35S, CaMV 35S promoter (0.84 kb); Bar, bar gene (0.55 kb); Tnos, nopaline synthase terminator (0.25 kb); MAR, 5'-matrix attachment region of chicken lysozyme gene (1.3 kb).

Figure 2

Detection of LTB, CTB and bar genes and phosphoacetyl transferase (PAT) in putative transgenic plant leaf tissues. (a) PCR amplification of LTB (217 bp), CTB (292 bp) and bar (302 bp) from transgenic rice genomic DNA. Lane 1 is the size marker. Lane 2 is the binary vector (pMJ103-LTB-CTB). Lane 3 is the wild-type. Lanes 4–28 are transgenic rice plants. (b) Lateral flow assay using the Trait LL lateral flow test kit (Strategic diagnostics). Lane 1 is the wild-type. Lanes 2–26 are transgenic rice plants. (c) Southern hybridization analysis of chromosomal DNA in transgenic rice. Genomic DNA from wild-type untransformed or independent transgenic rice T₀ lines carrying the pMJ103-LTB-CTB construct was hybridized with a probe specific for the LTB coding region. A total of 10 μg of total leaf genomic DNA from transgenic rice plants was digested with XhoI, which cuts at a single site within the binary vector pMJ103-LTB-CTB. (d) LTB and CTB gene transcripts in transgenic plants were detected by RT-PCR. Plant RNA was isolated from selected transgenic plant rice seeds and RT-PCR was performed using a primer pair that specifically amplified 107- and 111-bp DNA fragments of the LTB and CTB genes (lanes 2 to 8). Lane 1 shows the RNA of non-transgenic plants used as a negative control.

Figure 3

Homozygosity of transgenic rice lines (LCI-7 and LCI-11) was analyzed by PCR using RP, LP, BP, and RB sequences. (a) Three primer combinations were used in LCI-7 (left) and LCI-11 (right): the first comprised gene-specific right and left primers (RP1 + LP1 and RP2 + LP2); the second comprised a gene-specific right primer and T-DNA right border primer (RP3 + BP1 and RP2 + BP2); the third comprised a gene-specific left primer and a T-DNA left border primer (LP1 + RB1 and LP2 + BP3). Amplicon sizes are indicated on the right (in kb). (b) T-DNA insertion on chromosome 12 in the LCI-11 rice line. The chromosome regions in which T-DNA insertions were found ranged from 798,649 to 798,672 bp in length.

Figure 4

Molecular analyses of transgenic plants by PCR and real-time PCR in the LCI-7 and LCI-11 lines (T ₃ , T ₄ and T ₅ generations). (a) Genomic DNA isolated from T₃ to T₅ transgenic plants of LCI-7 (left) and LCI-11 (right) lines was used and the bar, LTB, and CTB genes were amplified by PCR. Lane 1: 1 kb marker: Lane 2: wild-type rice: Lane 3, 4 and 5: T_3–5 transgenic rice. (b) The LTB, CTB, and bar gene, and Tnos, copy numbers were analyzed in the T₃, T₄ and T₅ generations of the LCI-11 rice line by real-time PCR.

Figure 5

Recombinant LTB and CTB protein expression by transgenic rice seeds. (a) G_M1-ganglioside receptor binding assay of LTB and CTB protein expression in transgenic rice. The relative amount of LTB and CTB proteins present in LCI-11 was estimated by standard curves of recombinant LTB and CTB proteins, respectively. Arrows indicate relative amount of LTB and CTB proteins in LCI-11. BSA, bovine serum albumin. (b) Western blot analysis of LTB and CTB expression by transgenic rice seeds using LTB- (left) and CTB- (right) specific antibodies, respectively. Lane 1: positive control. Lane 2: wild-type rice. Lane 3: transgenic rice. Arrows indicate LTB and CTB proteins.

Figure 6

Levels of LTB- and CTB-specific IgG and IgA in mouse serum and feces. (a) CTB- and LTB-specific systemic IgG and mucosal IgA in mouse serum following intraperitoneal injection of wild-type (WT) and LCI-11 seeds and Ed coli-expressed LTB and CTB proteins. (b) LTB- and CTB-specific systemic IgG and mucosal IgA in mouse serum fed orally with wild-type and transgenic rice seeds. PBS was used as the negative control. Western blot (inset) detection of LTB- and CTB-specific IgG and IgA in mice immunized orally with LCI-11. First lane, BSA; second lane, LTB or CTB proteins, respectively. (c) LTB-specific mucosal IgA levels in mouse feces fed orally with recombinant LTB protein, wild-type and transgenic rice seeds. PBS was used as the negative control.